The present invention relates to transgenic non-human animal models of basal cell carcinoma. More specifically, the present invention is directed to mouse models of basal cell carcinoma allowing for the characterization of the mechanism of the disease as well as for developing and testing potential treatments.
Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure.
Approximately 1,000,000 epithelial skin cancers are diagnosed in the United States each year1, and the great majority of these are basal cell carcinomas (BCCs). The pathogenesis of these tumors involves constitutive activation of the Sonic hedgehog (Shh) signaling pathway2-4. In many BCCs this can be attributed to loss-of-function mutations involving PTCH15,6, which encodes a SHH receptor and antagonist. However, the identity of the specific downstream effector in the Shh pathway leading to cancer development remains unknown.
It is now demonstrated that transgenic mice overexpressing the transcription factor Gli2 in cutaneous keratinocytes develop multiple skin tumors that are grossly indistinguishable from human BCCs. These tumors express the same protein markers as human BCCs and exhibit strikingly elevated levels of Ptch1 and Gli1, a hallmark of human BCCs but not squamous tumors. These results establish Gli2 as a potent oncogene in skin and suggest a pivotal role for this transcription factor in the development of BCC, the most common cancer in humans.
With the development of transgenic animal models for basal cell carcinoma potential treatments including effective drug therapies can now be developed and tested.
The present invention provides transgenic non-human models of basal cell carcinoma and more specifically transgenic mouse models of basal cell carcinoma which allows for the characterization of the disease as well as for providing a system for the development and testing of potential treatments. In particular the invention provides transgenic mice overexpressing the transcription factor Gli2 in cutaneous keratinocytes which leads to the development of multiple skin tumors that are grossly indistinguishable from human BCCs. The present invention also provides methods for the production of non-human transgenic animal models of basal cell carcinoma as well as methods for testing compounds for an effect on basal cell carcinoma. The invention also encompasses isolated cells from the transgenic animal models as well as isolated eggs of the transgenic animals.
In accordance with the present invention there is provided a transgenic non-human animal model for basal cell carcinoma.
In accordance with another aspect of the present invention is a transgenic mouse model for basal cell carcinoma. The basal cell carcinoma as developed by the transgenic models physiologically resembles that of human basal cell carcinoma.
According to an aspect of the invention is a transgenic mouse model characterized by having a great similarity to several physiological conditions existing in naturally occurring human basal cell carcinoma, based on the expression of keratin 17 (K17), Bcl-2, keratin K5 and the non-expression of keratin K1 and keratin K6 as well as on histological analysis. Furthermore, the basal cell carcinoma tumors developed by the transgenic mice also express the same protein markers as human basal cell carcinomas as well as elevated levels of Ptch1 and Gli1.
According to another aspect of the invention is a transgenic mouse whose genome contains at least one copy of a Gli2 nucleotide sequence wherein said mouse develops basal cell carcinoma. The nucleotide sequence may be genomic DNA, cDNA or RNA.
According to a further aspect of the invention is a transgenic mouse whose somatic and germ cells comprise a Gli2 nucleotide sequence, wherein the expression of said sequence leads to the development of basal cell carcinoma.
In accordance with a first embodiment of the invention, a construct comprising a full-length mouse Gli2 cDNA with an amino-terminal FLAG tag under the control of a bovine keratin 5 (K5) promoter and also containing a rabbit -globin intron 2 sequence and an SV40 polyA signal was made. The construct was then introduced into mouse embryos using standard techniques such as microinjection. Founders were identified and any skin tumors identified and biologically characterized.
In accordance with a further embodiment of the invention, a binary transgenic system was utilized to target Gli2 overexpression in keratinocytes. In this approach, a Gli2 transgene was placed downstream of a xcex2geo (lacZ/neomycin) cassette flanked by a pair of loxP sites. Upstream of the xcex2geo cassette was placed a strong ubiquitous promoter such as the xcex2actin promoter. This construct was used to create Z/AP-Gli2 mice in which lacZ expression was driven in all cells. In this system, the Gli2 transgene is activated only when the xcex2geo cassette is excised by Cre recombination. For Gli2 overexpression, the Z/AP-Gli2 mice were crossed with a K5-Cre transgenic line producing K5-Cre:Z/AP-Gli2 mice having a high incidence of basal cell carcinoma. Furthermore, in initial experiments, Cre was electroporated into ES cell clones containing epitope tagged Gli2 or Gli2xcex94N and chimeric mice generated by aggregration of ES cells with morula stage mouse embryos. Z/AP-Gli2 and Z/AP- Gli2xcex94N mouse lines were established after germline transmission.
Animal cells can be isolated from the transgenic mice or prepared using the same constructs with standard techniques such as lipofection or electroporation. The transgenic animals or animal cells may then used to screen for compounds altering the pathological course of basal cell carcinoma leading to the development of pharmaceutical therapeutic approaches for treating the disease.
In accordance with a further aspect of the present invention is a transgenic mouse whose somatic and germ cells comprise a nucleic acid construct wherein the construct comprises a nucleic acid encoding Gli2 under the control of a suitable promoter and wherein the construct is transcribed. The construct may additionally contain any suitable polyA elements as well as a suitable regulatory element in order to enhance the expression of the Gli2 transgene.
In one embodiment of the invention the nucleic acid encoding Gli2 is a full length mouse cDNA with an amino-terminal FLAG tag operatively linked to a bovine keratin 5 (K5) promoter. In a further embodiment of the invention such construct further comprises a rabbit -globin intron 2 sequence and an SV40 polyA signal.
In another embodiment of the invention the nucleic acid encodes amino acids 280-1544 of Gli2 (herein referred to as Gli2xcex94N). In a further embodiment, the nucleic acid of the invention encodes Gli2 or a fragment or variant thereof which leads to Gli2 expression.
In accordance with still a further aspect of the present invention is a transgenic mouse whose somatic and germ cells comprise a nucleic acid construct wherein the construct comprises a full-length mouse Gli2 cDNA or variant thereof with an amino-terminal FLAG tag operatively linked to a bovine keratin 5 (K5) promoter and also containing a rabbit -globin intron 2 sequence and an SV40 polyA signal, and wherein the nucleic acid is expressed in skin cells of the transgenic mouse.
In accordance with another aspect of the present invention is a transgenic mouse whose somatic and germ cells comprise a nucleic acid construct wherein the construct comprises a full-length mouse Gli2 cDNA or variant thereof operatively linked to a xcex2actin promoter. The Gli2 transgene is activated by Cre recombination leading to expression of the Gli2 nucleic acid sequence in skin cells of the transgenic mouse such that the mouse develops basal cell carcinoma.
In accordance with still a further aspect of the present invention is a transgenic mouse whose somatic and germ cells comprise a nucleic acid construct wherein the construct comprises a full-length mouse Gli2 cDNA or variant thereof operatively linked to a bovine keratin 5 (K5) promoter and also containing a rabbit -globin intron 2 sequence and an SV40 polyA signal, and wherein the nucleic acid is over expressed in skin cells of the transgenic mouse such that the transgenic mouse develops basal cell carcinoma.
In yet further aspects, the invention features methods for using the non-human transgenic animals, cells and cell lines of the invention for investigating molecular and cellular mechanisms of Gli2 mediated pathogenesis leading to the development of basal cell carcinoma.
In accordance with yet a further embodiment of the present invention is a method of testing compounds for an effect on basal cell carcinoma, the method comprising:
administering the compound to be tested to a transgenic mouse whose genome comprises a nucleic acid construct, wherein the construct comprises nucleic acid encoding a Gli2 oncogene or variant thereof operably linked to a suitable promoter and wherein the transgenic mouse develops basal cell carcinoma; and
comparing one or more characteristics of the basal cell carcinoma tumors in the transgenic mouse to which the compound was administered with the same one or more characteristics of the tumors in the transgenic mouse to which the compound has not been administered, wherein a difference in one or more of the one or more characteristics indicates that the compound has an effect on basal cell carcinoma.
In accordance with still another embodiment of the present invention there is provided a method of identifying markers associated with basal cell carcinoma, the method comprising:
comparing the presence, absence or level of expression of genes in skin tissue from a transgenic mouse and skin tissue from a second mouse, wherein the genome of the transgenic mouse comprises a nucleic acid construct and the genome of the second mouse does not comprise the nucleic acid construct, wherein the construct comprises nucleic acid encoding a Gli2 oncogene or variant thereof operably linked to a suitable promoter, and wherein the nucleic acid is expressed in the skin cells of the transgenic mouse such that the transgenic mouse develops basal cell carcinoma,
wherein the difference between the transgenic mouse and the second mouse in the presence, absence or level of expression of a gene indicates that the expression of the gene is a marker associated with basal cell carcinoma.
In accordance with a further embodiment of the present invention are in vitro cell culture models for developing methods of treatment including the identification of therapeutically active agents for basal cell carcinoma, wherein the cells contain a nucleic acid construct comprising a nucleic acid encoding a Gli2 oncogene or variant thereof under the control of a suitable promoter and wherein the nucleic acid is expressed in the cells. Therapeutically active agents may include for example but are not limited to small molecule drugs including peptides and peptidomimetic drugs.
In accordance with a further embodiment of the present invention is a transgenic cell comprising a nucleic acid encoding a Gli2 oncogene or variant thereof operably linked to a suitable promoter and wherein the nucleic acid is expressed in the transgenic cell.
In accordance with yet another aspect of the present invention is a transgenic cell comprising a nucleic acid construct wherein the construct comprises a full-length Gli2 cDNA operatively linked to a bovine keratin 5 (K5) promoter and wherein the nucleic acid is expressed in the cells. The construct may further comprise a regulatory element to enhance expression of the transgene such as a rabbit -globin intron 2 sequence and also may contain a suitable polyA element such as a SV40 polyA signal. Such transgenic cells may be used in various assays to screen for compounds which are therapeutically effective against the disease.
In accordance with a further aspect of the present invention is a plasmid insert comprising a nucleic acid encoding a Gli2 oncogene or variant thereof under the control of a suitable promoter. Such plasmid insert being insertable in a mammalian genome in order to over express Gli2 in the desired cells, wherein such overexpression leads to the development of basal cell carcinoma. Preferably, the insert comprises a full-length Gli2 cDNA under the control of a keratin 5 (K5) promoter.
In accordance with another aspect of the present invention is a transgene construct comprising a nucleic acid encoding a Gli2 oncogene downstream of a xcex2geo cassette flanked by a pair of loxP sites. Downstream of the xcex2geo cassette are polyadenylation sites and upstream of the xcex2geo cassette is a ubiquitous promoter. The transgene is activated by excision of the xcex2geo cassette by Cre recombination.
In accordance with yet a further aspect of the present invention is the use of a Gli2 oncogene or variant thereof in a mammal in order to stimulate basal cell carcinoma development in said mammal.
In accordance with yet a further aspect of the present invention is the use of a Gli2 oncogene or variant thereof in a mammal in order to stimulate Shh target genes in said mammal.
In accordance with still a further aspect of the invention is the use of the Gli2 oncogene or variant thereof in a construct, such construct being insertable into a mammalian genome, wherein the Gli2 oncogene is expressible and stimulates basal cell carcinoma development.